Connecting TDP-43 Pathology to the Molecular Profiles of Neurodegeneration

TDP-43致病痴呆的分子水平与TDP-43病理生物学的关系 在之前的合作[1]中，Phatnani博士和Gale-Hammell博士使用一个大型ALS患者测序联盟来揭示这种TDP-43相关疾病患者额叶和运动皮质中主要的分子通路改变。在这项工作中，ALS皮质样本可以根据其转录谱是否显示出氧化应激、反转录转座子沉默或神经炎症的特征而被区分为3个不同的组。这些患者的组织学染色显示TDP-43蛋白病变与后两组患者密切相关，尽管TDP-43蛋白聚集体在逆转录转座子表达亚群中占主导地位。这笔赠款将建立在我们之前工作的基础上，以解决类似的TDP-43依赖的病理机制是否涉及与TDP-43功能障碍相关的常见痴呆，特别是伴有TDP-43蛋白病的额颞痴呆(FTD-TDP)和阿尔茨海默病(AD)。我们的假设是，反转录转座子在一定程度上导致了与FTD-TDP和AD的TDP-43功能障碍相关的毒性，我们将在这项提案中进行最后的测试。我们收集了200多名FTD-TDP或AD患者的大量组织样本，并进行了广泛的临床病理特征分析。这些样本的图谱将被用于指导TDP-43在FTD和AD中的病理机制的深入机制研究。 -- 目的1：TDP-43蛋白病变的细胞和组织特异性背景如何影响其在FTD和AD中的影响？我们将使用200名FTLD-TDP和AD患者死后皮质组织的多重免疫染色数据，以显示TDP-43病理特征，并在年龄匹配的猝死对照组中显示。我们将获得多个独立因素的高空间分辨率IHC图像，包括病理标记物(pTDP43和ptau)、细胞类型标记物以及我们先前工作中发现的与TDP-43功能障碍一致的通路成分，如炎症标记物(IBA1、TREM2)和反转录转座子蛋白(HERVK-env和L1HS-orf1)。在同一组织的相邻切片上，我们将使用空间转录学(ST)来定义TDP-43蛋白病的细胞类型特定和上下文特定影响的高分辨率转录图谱。这些结合的分子图谱将使我们能够直接评估TDP-43功能障碍对细胞类型特异性和蛋白质病近端效应的贡献。 -- 目的2：主动反转录转座子是简单地报告TDP-43功能障碍，还是对FTD和AD的细胞毒性有贡献？我们将获得FTD-TDP和AD患者样本中单个表达的反转录转座子的长读PacBio测序，以强有力地确定两种痴呆中特定表达的基因组反转录转座子座位，以及它们产生功能蛋白的相对能力。然后，我们将使用CRISPRa激活上面在iPS分化的皮质神经元中识别的单个反转录转座子，以测试相对的细胞毒性。在相反的实验中，我们将使用CRISPRi来抑制具有TDP-43功能障碍的iPS神经元中上述单个反转录转座子的活性，以测试其相对缓解TDP-43介导的神经毒性的能力。 -- 目的3：依赖TDP-43的反转录转座子能否促进星形胶质细胞和小胶质细胞的激活？在NYGC ALS Consortium患者中，我们之前在有TDP-43蛋白病变和激活的小胶质细胞证据的样本中看到了天然免疫信号通路的激活。此外，先前已经证明反转录转座子在其他情况下激活了天然免疫通路。这一目标将确定依赖TDP-43的逆转座子是否足以在FTD-TDP和AD的细胞模型中诱导邻近神经胶质细胞的激活。我们将在iPS来源的皮质神经元中表达单个反转录转座子，并在3D有机共培养系统中测试相邻星形胶质细胞和小胶质细胞的激活情况。在反向实验中，我们将在胶质细胞类型中表达TDP-43依赖的反转录转座子，以测试反转录转座子是否更有效地激活星形胶质细胞和小胶质细胞中的天然免疫信号通路，从而分泌神经毒性因子。

Connecting TDP-43 Pathobiology to the Molecular Profiles of TDP-43 Driven Common Dementias In previous collaborative work[1], Dr. Phatnani and Dr. Gale-Hammell used a large ALS patient sequencing consortium to uncover the predominant molecular pathways that are altered in the frontal and motor cortex of patients with this TDP-43 associated disease. In this work, ALS cortex samples could be differentiated into 3 distinct groups based upon whether their transcriptional profiles showed hallmarks of: oxidative stress, retrotransposon de-silencing, or neuroinflammation. Histological staining of tissues from these same patients showed tight links between TDP-43 proteinopathy and the latter two groups of patients, though TDP-43 protein aggregates were most predominant in the retrotransposon expressing subset. This grant will build on our prior work to address whether similar TDP-43 dependent pathomechanisms are involved in common dementias associated with TDP-43 dysfunction, specifically frontotemporal dementia with TDP-43 proteinopathy (FTD-TDP) and Alzheimer’s Disease (AD). Our hypothesis is that retrotransposons contribute in part to the toxicity associated with TDP-43 dysfunction in FTD-TDP and AD, which we will conclusively test in this proposal. We have amassed a large collection of tissue samples from over 200 patients with either FTD-TDP or AD, and with extensive clincopathological characterization. The profiles from these samples will be used to guide deep mechanistic studies into the pathomechanisms of TDP-43 in FTD and AD.   Aim 1: How does the cell- and tissue-specific context of TDP-43 proteinopathy affect its impact in FTD and AD? We will use multiplexed immunostaining data of postmortem cortical tissue from 200 FTLD-TDP and AD patients selected to show signatures of TDP-43 pathology, and in age-matched sudden death controls. We will obtain high spatial resolution IHC images of multiple independent factors, including pathological markers (pTDP43 and pTau), markers of cell type, and components of pathways identified to be coincident with TDP-43 dysfunction in our previous work such as inflammatory markers (IBA1, TREM2) and retrotransposon proteins (HERVK-env and L1HS-orf1). At adjacent slices from the same tissue, we will use Spatial Transcriptomics (ST) to define a high-resolution transcriptomic atlas of cell-type specific and context-specific impacts of TDP-43 proteinopathy. These combined molecular profiles will allow us to directly assess the contribution of TDP-43 dysfunction to cell-type specific and proteinopathy-proximal effects.   Aim 2: Do active retrotransposons simply report on TDP-43 dysfunction or contribute to cellular toxicity in FTD and AD? We will obtain long read PacBio sequencing of individual expressed retrotransposons in FTD-TDP and AD patient samples to robustly determine the specific expressed genomic retrotransposon loci in both dementias, and their relative ability to produce functional proteins. We will then use CRISPRa to activate individual retrotransposons identified above in iPS differentiated cortical neurons to test for relative cellular toxicity. In the converse experiment, we will use CRISPRi to inhibit the activity of individual retrotransposons identified above in iPS neurons with TDP-43 dysfunction to test for the relative ability to alleviate TDP-43 mediated neurotoxicity.   Aim 3: Can TDP-43 dependent retrotransposons contribute to activation of astrocytes and microglia? In the NYGC ALS Consortium patients, we previously saw activation of innate immune signaling pathways in samples with evidence of TDP-43 proteinopathy and activated microglia.  Moreover, retrotransposons have previously been shown to activate innate immune pathways in other contexts. This aim will establish whether TDP-43 dependent retrotransposons are sufficient to induce activation of adjacent glial cells in cellular models of FTD-TDP and AD. We will express individual retrotransposons in iPS derived cortical neurons and test for the activation of adjacent astrocytes and microglia in a 3D organoid co-culture system. In the converse experiment, we will express TDP-43 dependent retrotransposons in glial cell types, to test whether retrotransposons more potently activate innate immune signaling pathways in astrocytes and microglia, which subsequently secrete neurotoxic factors.